Copyright  2001 by the Genetics Society of America

Isolation and Characterization of the Xanthine Dehydrogenase Gene

of the Mediterranean Fruit Fly, Ceratitis capitata

R. J. Pitts and L. J. Zwiebel

Department of Biological Sciences, Vanderbilt University, Nashville, Tennessee 37235
Manuscript received February 1, 2001
Accepted for publication May 23, 2001

ABSTRACT
Xanthine dehydrogenase (XDH) is a member of the molybdenum hydroxylase family of enzymes
catalyzing the oxidation of hypoxanthine and xanthine to uric acid. The enzyme is also required for the
production of one of the major Drosophila eye pigments, drosopterin. The XDH gene has been isolated
in many species representing a broad cross section of the major groups of living organisms, including the
cDNA encoding XDH from the Mediterranean fruit fly Ceratitis capitata (CcXDH) described here. CcXDH
is closely related to other insect XDHs and is able to rescue the phenotype of the Drosophila melanogaster
XDH mutant, rosy, in germline transformation experiments. A previously identified medfly mutant, termed
rosy, whose phenotype is suggestive of a disruption in XDH function, has been examined for possible
mutations in the XDH gene. However, we find no direct evidence that a mutation in the CcXDH gene
or that a reduction in the CcXDH enzyme activity is present in rosy medflies. Conclusive studies of the
nature of the medfly rosy mutant will require rescue by germline transformation of mutant medflies.

T HE development of improved control strategies for

insects that act as biological pests and disease vec-
tors is vitally important for the prevention of the spread
of human disease and for the alleviation of damage
to economically important domestic animals and plant
species. One of the most notorious agricultural pests is
the Mediterranean fruit fly (medfly), Ceratitis capitata.
The medfly has migrated from its origins in Africa
throughout the Mediterranean region and into the
Americas within the last 100 years (Haymer et al. 1997;
Malacrida et al. 1998; Davies et al. 1999) and is respon-
sible for the global annual loss of billions of dollars in
fruit crop production. As a consequence, medflies are
the subject of intense control efforts in many parts of
the world, including Central America, Europe, and the
United States (Kahn et al. 1990; Barinaga 1991; Carey
1991). The medfly is particularly destructive because it
has a very wide host plant range, being able to infect
some 200 fruit varieties, and because females puncture
the fruit when laying eggs, allowing for larval and op-
portunistic microbial invasion (Saul 1986; Robinson
1989). It is hoped that a detailed understanding of the
biology of the medfly will eventually lead to the develop-
ment of the tools needed to effectively manage its popu-
lations. Germline transformation is one of the tools
likely to contribute to the design of novel and effective
control strategies.
To facilitate germline transformation, selectable or
Corresponding author: L. J. Zwiebel, Department of Biological Sci-
ences, Box 82, Station B, Vanderbilt University, Nashville, TN 37235.
E-mail: l.zwiebel@vanderbilt.edu
Genetics 158: 1645–1655 (August 2001)
visible phenotypic markers are required to separate
transformants from nontransformants. Many such visi-
ble markers have been used to great effect in the widely
studied insect model system, Drosophila melanogaster. Cur-
rently, the two genetic markers most routinely used are
the eye color genes white and rosy (Ashburner 1989).
In Drosophila, white encodes an ABC family transporter
that is responsible for import of eye pigments into pho-
toreceptor cells (reviewed in Higgins 1992) while rosy
encodes xanthine dehydrogenase (XDH), a member
of the molybdenum-containing hydroxylase family of
enzymes (Keith et al. 1987). The Drosophila rosy/XDH
gene enzyme system has been studied in great detail,
and it has long been established that XDH is required
for the production of drosopterin eye pigment as well
as for the conversion of purines into uric acid (reviewed
in Chovnick et al. 1990; Wootton et al. 1991; Hille and
Nishino 1995). The use of these genes in Drosophila
transformation experiments has led to the suggestion
of their similar application as phenotypic markers for
germline transformation in the medfly and other insects
(Zwiebel et al. 1995). Several medfly eye color mutants
have been described, including white, rosy, light eye, and
Purple eye (reviewed in Saul 1985, 1986) and, to date,
white is the only one whose gene has been fully isolated
from medfly (Zwiebel et al. 1995; Gomulski et al. 2001).
As in Drosophila, the medfly white mutants completely
lack eye pigmentation (Rossler and Koltin 1976; Ros-
sler and Rosenthal 1992). Significantly, the medfly
white gene is homologous to Drosophila white, and two
naturally occurring mutations have been defined at the
DNA level (Zwiebel et al. 1995; Gomulski et al. 2001).
1646 R. J. Pitts and L. J. Zwiebel
Indeed, partial phenotypic rescue of white mutants has
been successfully carried out in medfly using the medfly
white cDNA as a dominant marker for two transposon-
based transformation vectors, Minos and piggyBac
(Loukeris et al. 1995; Handler et al. 1998).
As is the case in Drosophila, the characterization of
multiple molecularly defined phenotypic markers for
transformation in the medfly will facilitate increased
flexibility among different applications. As part of the
effort to expand the range of germline transformation
systems in this important agricultural pest insect, we
have undertaken the isolation and characterization of
the medfly XDH gene. By analogy to the white gene,
Ceratitis XDH (CcXDH) has the potential to be used as
an additional phenotypic marker, but only if a corre-
sponding medfly XDH mutant with an easily distinguish-
able phenotype can be identified. A previous study has
isolated and characterized a C. capitata mutant, termed
rosy, which phenotypically displays burgundy eyes as well
as a sensitivity to purine-supplemented media (Saul
1982), two phenotypes that are characteristic of many
of the rosy alleles of D. melanogaster (reviewed in Chov-
nick et al. 1990). This would suggest that the molecular
basis for the rosy mutant may be a defect in the CcXDH
gene. Alternatively, these phenotypes could be the result
of a mutation in a gene affecting CcXDH function be-
cause the enzyme requires the actions of several cofac-
tors and gene products for its normal function in Dro-
sophila (Hughes et al. 1992; Hughes 1992). For
example, the product of the ma-l gene is required for
XDH activity (Forrest et al. 1956; Glassman and
Mitchell 1959) where it is apparently responsible for
the ability of XDH and other enzymes to incorporate
sulfur (Wahl et al. 1982). Mutations in ma-l and other
loci in Drosophila, including low xanthine dehydrogenase
(lxd) and cinnamon (cin), also display a rosy eye pheno-
type and have a corresponding low level of XDH activity
(reviewed in Kamdar et al. 1997). It will be critical to
elucidate whether a lesion in XDH causes the medfly
rosy mutant because it has the potential to be used in
germline transformation.
To directly examine this issue we have cloned the
full-length medfly CcXDH cDNA and have tested the
hypothesis that the medfly rosy phenotype is caused by
a defect in CcXDH. Our studies demonstrate that the
CcXDH gene is capable of functionally rescuing the
Drosophila XDH rosy mutant and may therefore be use-
ful as a marker for medfly germline transformation in
a CcXDH mutant background. However, we find no
evidence for a defect in the XDH gene in the medfly
rosy mutant at the levels of DNA, RNA, or enzyme activity
in vitro. Final resolution of this question requires the
use of CcXDH cDNA in an attempt to rescue the medfly
rosy mutant by germline transformation. We are pre-
vented from conducting such studies due to the absolute
quarantine against live medflies that is currently in place
in the continental United States.
MATERIALS AND METHODS
Medfly strains: Two independently derived medfly labora-
tory strains were used in this study: Benakeion, which is associ-
ated with the XDH⫹ allele and has wild-type eye color, and the
rosy eye mutant strain, in the Wiedemann genetic background.
The wild-type eye color strain Benakeion was originally estab-
lished in the laboratory by P. A. Mourikis (Benakeion Institute
of Phytopathology, Athens, Greece) with flies from the South-
ern Peloponnese (Greece) and Palermo (Italy; Rina and
Savakis 1991). The rosy strain was established in the laboratory
of Stephen Saul (Saul 1982).
C. capitata XDH cloning: CcXDH clones were isolated using
degenerate PCR primers designed using the medfly codon
bias values (Nakamura et al. 2000) against conserved domains
of rat XDH and two available insect sequences, D. melanogaster
and C. vicina. The primers were LJZVI 5⬘-ACI GCI TTY CGY
GGY TTY GGI GGI CCW CAR GGI ATG-3⬘ (corresponding
to the amino acid sequence TAFRGFGGPQGM) and LJZVIII
5⬘-YTG ICC RAT RTC IGC DGG RTT RAA IGA IGA ICC-3⬘
(corresponding to the amino acids QGSSLNPAIDIGQ). PCR
generated an 860-bp product that was TOPO TA cloned (In-
vitrogen, San Diego) and sequenced to verify that it was XDH
from medfly. This sequence corresponds to amino acids start-
ing from position 1155 of the full CcXDH peptide VGDD…etc.
The 860-bp fragment was radiolabeled with [dATP]␣32P and
used to isolate both genomic and cDNA clones from ␭-phage
libraries (Sambrook et al. 1989). Genomic clone 623 remains
uncharacterized while ␭zap (Stratagene, La Jolla, CA) cDNA
clone 114-1 was subcloned into the KpnI and SalI restriction
sites of vector pSP73 (Promega, Madison, WI).
Poly(A)⫹ RNA was isolated from adult medflies (C. capitata)
and used to synthesize double-stranded cDNA followed by
adapter ligation using the Marathon cDNA amplification kit pro-
tocol (CLONTECH, Palo Alto, CA). Adapter oligonucleotide
primer AP2 5⬘-ACT CAC TAT AGG GCT CGA GCG GC-3⬘ was
used in combination with oligonucleotide primer XDHRACE
5⬘-AGC ATA CAA CGC ACG GGT CTT C-3⬘ to PCR amplify
the 5⬘ end of the XDH cDNA from the medfly rapid amplifica-
tion of cDNA ends (RACE) library under the following condi-
tions. A premix of 17.5 ␮l 10⫻ Clontech RACE PCR buffer,
14 ␮l [10 mm] dNTP, 3.5 ␮l of Advantage Taq polymerase,
and 119 ␮l dH2O was mixed and kept on ice. Each reaction
contained 21.5 ␮l of premix, 2.5 ␮l of a 1:200 dilution of
adapter ligated RACE library, and 0.5 ␮l of each [10 ␮m]
primer. A positive control reaction containing 2.5 ␮l of control
cDNA and 0.5 ␮l of oligos AP1 5⬘-CCA TCC TAA TAC GAC
TCA CTA TAG GGC-3⬘ and TFR3⬘ 5⬘-ATT TCG GGA ATG
CTG AGA AAA CAG ACA GA-3⬘ produced the expected 2.9-kb
product. Negative control reactions containing single primers
produced no products. Reactions were carried out in a Perkin-
Elmer (Norwalk, CT) 9700 thermal cycler as follows: 94⬚ for
2 min; 5 cycles of 94⬚ for 5 sec and 72⬚ for 4 min; 5 cycles of
94⬚ for 5 sec and 70⬚ for 4 min; and 25 cycles of 94⬚ for 5 sec
and 68⬚ for 4 min.
Digesting the 5⬘ RACE subclone with XhoI/BbsI restriction
endonucleases generated the full-length medfly cDNA clone
(2.8 kb). This fragment was ligated into a SalI/BbsI-digested
vector containing the 3⬘ end of XDH. The full-length cDNA,
pSP73:CcXDH, was sequenced in an ABI377 automated se-
quencer as described (Perkin-Elmer). CcXDH was conceptu-
ally translated and alignments with similar peptides were per-
formed using CLUSTAL W software (Thompson et al. 1994).
The PAUP software package (Swofford 1991) was used to
determine the phylogenetic placement of our medfly XDH
sequence in relation to those of the full-length XDH genes
previously sequenced and analyzed from other insect taxa
(Komoto et al. 1999). These included sequences from two
 XDH cDNA of C. capitata
different XDH loci from Bombyx mori as well as single copies
from the drosophilid species D. melanogaster, D. pseudoobscura,
and D. subobscura, and from the calliphorid Calliphora vicina.
B. mori is the only insect reported to possess more than one
XDH gene, each apparently serving a different current func-
tion (Yasukochi et al. 1998; Komoto et al. 1999). Our initial
analyses used mouse XDH as an outgroup, while later analyses
deleted mouse and used Bombyx sequences instead. Equally
weighted XDH data sets were analyzed as both nucleotide and
amino acid sequences using maximum parsimony, neighbor
joining, and maximum likelihood methods. The bootstrap
(Felsenstein 1985) was used to assess statistical support for
relationships via branch and bound analysis of 100 pseudorep-
licated data sets.
D. melanogaster transformation: The Drosophila P-element
vector, pP{CaSpeR-hs/act}(GenBank accession no. U60735;
for vector map see http://www-hhmi.genetics.utah.edu/thum-
mel/pelement.html), mini prep DNA (1 ␮g) was digested with
NotI and BamHI and pSP73:CcXDH mini prep DNA (1 ␮g)
with NotI and BglII. Digests were run on 0.7% agarose (TAE)
gels. The 9.2-kb vector and 4.9-kb XDH insert sequences were
excised from the gel with sterile razor blades and DNA was
isolated from the gel slices using the QIAquick gel extraction
kit (QIAGEN, Valencia, CA). Ligations were set up as follows
and were allowed to proceed overnight at 16⬚: 1 ␮l pP{CaSpeR-
hs/act} fragment; 1 ␮l or 2 ␮l of pSP73:XDH NotI/BglII frag-
ment; 1 ␮l T4 ligase buffer; 0.3 ␮l T4 ligase [400 units/␮l];
dH2O to 10 ␮l. Each ligation (2 ␮l) was transformed into XL1-
Blue competent cells (40 ␮l) by electroporation (2.5 kV).
Transformations were plated onto Luria broth (LB) ⫹ ampicil-
lin (50 ␮g/ml) agar and incubated overnight at 37⬚. Fresh
ampr colonies were picked with a sterile toothpick and used
to inoculate an overnight LB ⫹ ampicillin50 culture (3 ml).
Plasmid DNA was isolated by alkaline lysis mini prep and
digested with SalI to confirm XDH insertion.
A plasmid DNA prep (⬎100 ␮g) of one pP{CaSpeR-hs/
act}:CcXDH clone was sent to the laboratory of Nick Brown
who performed injections into D. melanogaster. This vector was
injected into true-breeding white, forked (wf ) embryos of D.
melanogaster (G0 generation). The injected adults were crossed
back to wf adults, and individual w⫹ G1 flies were crossed
with wf adults to establish a G2 stock. Nine individual G2
transformed lines were established (letters A through I). W ⫹
G2 virgin females were crossed with cn/cn (II); Ly ry506/TM3
Sb ry506 (III) males. W ⫹ G3 males with forked bristles and non-
Lyra wings (and therefore Sb ry506) were crossed to BcElp/CyO
(II); ry506/ry506 (III) virgin females. The G4 crosses were heat
shocked every day for 1 hr at 37⬚ until adult eclosion. G4
adults were scored for bristle type, wing type, and eye color.
All G4 stubble flies were therefore homozygous for ry506.
Genomic DNA analysis: Genomic DNA was isolated from
wild-type C. capitata (Benakeion) adults or rosy pupae (Wiede-
mann) according to the protocol of Ish-Horowicz for Drosoph-
ila (protocol 47 in Ashburner 1989). Southern blots were
carried out using Hybond N⫹ membrane according to the
manufacturer’s protocol (Amersham Pharmacia Biotech). A
full-length XDH cDNA fragment was used to probe blot under
low stringency conditions. PCR was performed as follows:
ⵑ100 ng of DNA, dNTPs [0.2 mm], 1⫻ buffer (Perkin-Elmer)
with MgCl2 [0.15 mm], Taq polymerase, primers [0.2 ␮m],
and dH2O to 25 ␮l. Primer pairs started at the 5⬘ end of the
gene and continued along the coding sequence to the 3⬘ end
with each product overlapping the previous product. In this
manner the entire coding region was examined. Primer com-
binations used for coverage were XDH for 5⬘-TAG ATA ACA
GAA GCA TTT GGA-3⬘ and Xex1R 5⬘-ACC TTT TTC CCA
TTG ACA AAA-3⬘ (223 bp); Xex2F 5⬘-TAT TGA TCC CAC
ACC CGA T-3⬘ and XDH7 5⬘-AGC AAA TCT GAA AGC TCC
1647
AC-3⬘ (686 bp); XDH3 5⬘-CAC CAG AAC TGC ATT TAA
AC-3⬘ and XDHRACE 5⬘-AGC ATA CAA CGC ACG GGT CTT
C-3⬘ (1863 bp); XDH5 5⬘-CAC CGC GAG ATA GTG ATG
AA-3⬘ and XDH1 5⬘-TTA CTT ATG CAC TCC TGC C-3⬘ (1245
bp); and XDH6 5⬘-CGT GCA TTA GGT ATA CCA AC-3⬘ and
XDH3⬘end 5⬘-TTT GGC CAA TCC AAT CAG TT-3⬘ (1016
bp). No differences were detectable between PCR product
sizes when 5 ␮l of each reaction were run side by side on 0.7%
agarose gels.
RNA Analysis: C. capitata total RNA was isolated from 1-day-
old pupae of the rosy (Wiedemann) mutant and from embryos,
pupae, and adults of wild type (Benakeion), using the RNeasy
RNA isolation kit (QIAGEN). RT-PCR was performed using
the Titan One-Tube RT-PCR kit (Roche Molecular Biochemi-
cals). The manufacturer’s protocol was followed except that
reactions were scaled down from 50 to 25 ␮l by using half the
amount of each reagent. About 0.5 ␮g of each RNA sample
and a 0.2-␮m final concentration of XDHleft and XDH6 prim-
ers were used for each reaction. First-strand synthesis was per-
formed at 50⬚ for 30 min. This step was followed immediately
by 10 cycles of 94⬚ for 30 sec, 53⬚ for 30 sec, and 68⬚ for 45
sec, and then 30 cycles of 94⬚ for 30 sec, 53⬚ for 30 sec, and
68⬚ for 45 sec ⫹ 5 sec per cycle. Reactions were concluded at
68⬚ for 7 min. Five microliters of each reaction was analyzed
on a 1.5% agarose gel.
XDH enzyme assay: Crude extracts were prepared by ho-
mogenizing single medfly pupae or five Drosophila adult flies
in 1.5 ml Eppendorf tubes in 80 ␮l cold buffer: 100 mm Tris,
1 mm EDTA, 0.5 mm NAD, and 0.05% 2-mercaptoethanol
(pH 7.5). Each homogenate was centrifuged for 5 min at
13,000 rpm and 4⬚. Supernatant was transferred to a new tube
and centrifuged as before. Protein concentrations of each
extract were measured using the bicinchoninic acid method
and according to the manufacturer’s protocol (Pierce Chemi-
cal). Five microliters of each extract were loaded onto cellulose
acetate gels that had been pretreated in a running buffer of
61.4 mm Tris, 4 mm EDTA, and 13.6 mm citric acid (pH 7.5).
Gels were run at 100 V for 20–30 min. Gels were stained in
Tris buffer with 1.4 mm hypoxanthine, 2.4 mm NAD, 0.4 mm
phanazine methosulfate (PMS), and 1.2 mm nitro blue tetrazo-
lium (NBT) (or xanthine:PMS:cytC or xanthine:NAD⫹).
RESULTS
C. capitata cDNA: The full-length CcXDH cDNA se-
quence is 4397 bp in length, including the 5⬘ and 3⬘
untranslated sequences (UTR; GenBank accession no.
AY014961). The coding region spans 4041 bp, with the
ATG translational start codon located at position 223
and the TGA stop codon located at position 4264. The
ATG at position 223 is presumed to be the correct trans-
lational start because it is the first methionine following
several cryptic stop codons in the 5⬘ UTR, including
one that is just eight codons upstream in the same read-
ing frame. Furthermore, this start site facilitates the
longest possible open reading frame that is consistent
with the sizes of closely related XDH sequences while
the next potential start point lies 65 codons downstream.
Within the 3⬘ UTR, a potential polyadenylation signal
sequence, AATACA, precedes the observed polyadenyl-
ation site of the cDNA by 20 bp.
The cDNA encodes a peptide of 1347 amino acids
when conceptually translated (Figure 1A). Highly con-
 1648
R. J. Pitts and L. J. Zwiebel

Figure 1.—Medfly XDH gene. (A) Structure of the CcXDH gene. Boxes represent exons 1 through 6 of indicated size and thin lines denote introns A through G.
Conserved functional areas of enzyme are 2Fe/2S, iron-sulfur domains (stippled box); NAD⫹, nucleotide binding site (shaded box); and MoCo, molybdenum cofactor
(solid boxes). (B) Partial amino acid sequence alignments of highly conserved domains. Identical residues are boxed while conserved residues are shaded. Asterisks denote
the eight cysteines and the single tyrosine implicated in XDH function.
 XDH cDNA of C. capitata
served structural genes for XDH have been cloned from
a wide range of species including bacteria, fungi, plants,
insects, birds, and mammals (Keith et al. 1987; Houde
et al. 1989; Riley 1989; Amaya et al. 1990; Terao et al.
1992; Ichida et al. 1993; Glatigny and Scazzocchio
1995; Sato et al. 1995; Berglund et al. 1996; Comeron
and Aguade 1996). Of these, CcXDH is most similar in
size and sequence to other insect XDH peptides. Over-
all, it is 74–75% identical to XDH peptides from C.
vicina, D. melanogaster, D. pseudoobscura, and D. subob-
scura, all of which are dipteran flies. The medfly peptide
is somewhat less similar to the two XDH peptides identi-
fied in the silkworm moth, B. mori (order Lepidoptera),
displaying 58 and 52% identity to the B. mori 1 and B.
mori 2 peptides, respectively. A partial alignment (Figure
1B) of the seven peptides from the six available insect
species shows that XDH is highly conserved within the
regions thought to bind iron-sulfur (2Fe-2S), flavin ade-
nine dinucleotide, and molybdenum (MoCo) cofactors
(Hughes et al. 1992; Sato et al. 1995; Doyle et al. 1996).
Notably, there are eight completely conserved cysteine
residues, four in each iron-sulfur domain, that are likely
to participate in the binding of iron-sulfur cofactors and
a conserved tyrosine residue at position 407 that is likely
to bind NAD⫹ (asterisks, Figure 1B). The cysteine resi-
dues correspond positionally with cysteines of the alde-
hyde oxido-reductase (Mop) from Desulfovibrio gigas
that, on the basis of crystal structure analysis, have been
directly implicated in iron-sulfur binding (Romao et al.
1995; Romao and Huber 1997). The tyrosine corre-
sponds to the same residue of the chicken xanthine
dehydrogenase that is reported to participate in NAD
binding (Nishino and Nishino 1989).
Genomic DNA Southern blot analysis indicates that
CcXDH is a single copy gene in the medfly (Figure
4A), as is the case in Drosophila (Keith et al. 1987).
Furthermore, all phylogenetic analyses provide statisti-
cal corroboration of the findings of Komoto et al. (1999)
where Drosophila XDH sequences formed a monophy-
letic grouping within a more inclusive dipteran clade,
with the two Bombyx moth sequences being united in
a separate clade. While our medfly sequence was firmly
placed inside the dipteran clade, the relationships among
medfly, C. vicina, and Drosophila XDH sequences varied
1649
Figure 2.—XDH phylogram. Nucleo-
tide sequences of insect XDH genes were
aligned and phylogenetic relationships
were assigned as described in materials
and methods. Numbers indicate boot-
strap values. The XDH gene from Mus
musculus was used as an outgroup.
among analyses. Some estimates of dipteran phylogeny
suggest that the Tephritidae (including the medfly) are
more closely related to the Drosophilidae than either is
to the Calliphoridae (McAlpine 1989). However, more
recent estimates made by comparing the sequences of
two dipteran genes, glucose-6-phosphate dehydrogenase,
G6pdh (Soto-Adames et al. 1994), and white (Gomulski et
al. 2001), suggest that the Tephritidae are more closely
related to the Calliphoridae than to the Drosophilidae.
In this study, XDH from the calliphorid C. vicina was
observed to group with either the Drosophila or with C.
capitata in certain analyses. Nonetheless, the relationships
among these three lineages were not strongly supported
by bootstrap in any single analysis and are presented here
as unresolved (Figure 2).
PCR analysis indicates that at least four introns are
found within the CcXDH gene, and evidence is described
for a fifth. In these studies, genomic DNA was used as a
PCR template for a series of primers covering the XDH
coding region, and products that were larger than cDNA
control fragments were subcloned and sequenced. In this
manner, four small introns (introns 2 through 5) were
identified, ranging in length from 59 to 85 bp (Figure 1A
and Table 1). The positions of three of these introns, D,
F, and G (Komoto et al. 1999), are absolutely conserved
when compared with the positions of introns of other
insect species (Table 1), all of which are bounded by
gt-ag splice site consensus sequences (Breathnach and
Chambon 1981). We confirm the results of Tarrio et al.
(1998), who discovered that one of these four introns
(intron U in Figure 1A) is absent in all other reported
insect XDH genomic sequences (Tarrio et al. 1998). This
unique medfly intron does not correspond to the position
of intron E (Komoto et al. 1999) and it is believed to be
a duplication of intron D because their sequences are very
closely related in the medfly (Tarrio et al. 1998).
There are also considerable lines of evidence suggesting
the existence of a large first intron in medfly. Many of
the introns within insect XDH genes are positionally con-
served and all of the known insect XDH genes contain a
commonly located intron A as their first, and largest, in-
tron (Komoto et al. 1999). The size range of the first
intron varies from 815 bp in D. melanogaster to 15 kb in C.
vicina (Keith et al. 1987; Houde et al. 1989). Furthermore,
1650 R. J. Pitts and L. J. Zwiebel

TABLE 1
XDH gene intron sizes

Conserved intron positions

A B C D U E F G
C. cap ⬎5kb — — 65 82 — 85 59
C. vic 15kb — — — — — 67 51
D. mel 815 — — 281 — — — 65
D. pse 1024 — — 62 — — 67 67
D. sub 1535 — — 528 — — 62 68
B. mor1 4488 1198 75 77 — 897 454 2040
B. mor2 1398 395 455 76 — 165 1247 847
Conserved intron positions A through G are lettered according to Komoto et al. (1999). The size of each
intron is listed in base pairs except where indicated in kilobase pairs. Hyphens denote the lack of a particular
intron within a species. Intron U is unique to C. capitata (Tarrio et al. 1998).

Southern blots of medfly genomic DNA, probed with PCR indicating that the CcXDH transgene was both func-
products derived from cDNA spanning the putative splice tional and apparently able to fully complement the ry
site, hybridize with large molecular weight bands, indicat- mutant phenotype (Figure 3D). In contrast, the G4 stub-
ing an intron size of ⬎5 kb (data not shown), and several ble males, which do not carry the X-linked XDH trans-
attempts to PCR amplify the first intron region of the XDH gene and are also ry homozygotes, retained the rosy eye
gene from wild-type genomic DNA failed. Nonetheless, mutant phenotype under the same heat-shock condi-
genomic PCR reactions designed to amplify the exons tions and served as an internal negative control (Figure
immediately surrounding the putative intron site gave 3C). This experiment establishes the CcXDH as the func-
products of the expected length for cDNA and therefore tional ortholog of the Drosophila XDH.
indicate that these regions themselves are uninterrupted Medfly rosy mutant: To initially address the possibility
by introns (data not shown). that the medfly rosy mutant carries a defect in the XDH
Rescue of Drosophila rosy mutant: To demonstrate that gene, we carried out comparative Southern blot and
CcXDH encodes a functional xanthine dehydrogenase en- PCR analyses between genomic DNA prepared from
zyme in vivo, phenotypic rescue experiments were carried wild-type and rosy medflies. Inasmuch as identical bands
out using the well-established P-element transformation on Southern blots and identical PCR products were
protocols available in D. melanogaster (Rubin and Sprad-
ling 1982; Ashburner 1989). Prior to transformation,
and as an indication that the CcXDH cDNA could be
fully translated, a peptide of ⵑ150 kD corresponding
to the expected size for the full-length XDH monomer
(Edwards et al. 1977; Keith et al. 1987) was observed in
an in vitro rabbit reticulocyte translation system (data
not shown). The full-length CcXDH cDNA was subse-
quently cloned into the P-element transformation vec-
tor, pP{CaSpeR-hs/act}, which utilizes a white mini gene
for transformation selection, a 5⬘ heat-shock promoter
to drive expression of the insert, and 3⬘ Act 5C se-
quences to promote transcript stability. This construct
was introduced into the germline of white, forked recipi-
ent D. melanogaster flies that were then crossed into an
appropriate ry⫺ background. Ultimately, two trans-
formed lines, B and I, each having X chromosome inser-
tions, were chosen for detailed rescue analyses. The
results from one such experiment with line B are pre-
sented in Figure 3 and are representative of repeated
analyses with both lines B and I. In these studies, all G4 Figure 3.—Rescue of Drosophila rosy with CcXDH. Eye
females inherit the CcXDH transgene and those with color phenotypes of (A) Oregon-R (OR) wild type, (B) ry506
homozygous mutant, (C) G4 ry/ry males, and (D) G4 ry/ry
stubble bristles are homozygous for the ry mutation (see transgenic females. Arrows denote bristle phenotype: wild type
materials and methods). Under heat-shock condi- in A and B and Stubble in C and D, indicating the presence
tions, all these flies have wild-type eye pigmentation, of the ry balancer chromosome.
 XDH cDNA of C. capitata
Figure 4.—XDH gene analysis in medfly. (A) Southern blot
displaying single copy nature of medfly XDH gene. ⫹ and ry
indicate Benakeion and rosy genomic DNAs, respectively, while
numbers indicate sizes in kilobase pairs. Bands of 0.9 kb and
1.3 kb were expected for AccI digests while a band of 2.5 kb
was expected for AvaII digests. (B) RT-PCR analysis of RNA
isolated from three developmental stages of wild type and one
stage of the rosy mutant all show XDH transcript expression.
Oligonucleotide primers spanning intron F result in products
of 743 and 658 bp in genomic and cDNA controls, respectively.
observed in a series of side-by-side reactions covering
the entire coding region of the gene, these comparisons
show the medfly rosy XDH gene to be indistinguishable
from the wild-type gene (Figure 4A; PCR data not
shown). Furthermore, RT-PCR comparisons demon-
strated the presence of indistinguishable XDH tran-
scripts in RNA isolated from various life cycle stages of
wild-type and rosy medfly pupae, the only life cycle stage
of the rosy mutant at our disposal (Figure 4B). Finally,
because some of the primers used in these RT-PCR
studies were located near the 3⬘ end of the gene where
mRNA instability is likely to be the greatest, the rosy
mutant most likely produces a full-length transcript.
Given that the XDH gene of the medfly rosy mutant
was indistinguishable from the wild type at the DNA
and RNA levels within the limits of our studies, and in
the absence of appropriate antisera that might be used
for Western or immunohistochemical analyses, we
tested wild-type and rosy medflies for XDH enzyme activ-
ity using a cellulose acetate gel technique (Meera Khan
1971; Malacrida et al. 1992). In these studies, crude
extracts were prepared from wild-type and rosy mutant
pupae, subjected to cellulose acetate gel electrophore-
sis, and subsequently stained for XDH activity using
either xanthine or hypoxanthine as a reducing sub-
1651
strate. Figure 5A shows a typical gel from this study, in
which extracts from medfly rosy and wild-type pupae
appear to have similar levels of XDH activity. Impor-
tantly, when similar amounts of protein were loaded in
each lane, control extracts from D. melanogaster OR wild
type have high XDH activity, while extracts from ry506,
an XDH null mutant (Gelbart et al. 1974; Clark et al.
1986), lack any visible activity (Figure 5A). Replicate
experiments suggest that the difference in migration
observed in Figure 5A between wild-type and rosy medfly
extracts is the result of slight gel loading variation in the
absence of predefined sample wells. While no attempts
were made to quantify XDH activity assayed here, no
differences in enzyme activity between wild-type and rosy
medfly extracts were detected on cellulose acetate gels
using three different assay systems: hypoxanthine: NAD⫹:
PMS:NBT; xanthine:NAD⫹; or xanthine:PMS/cytC under
similar conditions.
DISCUSSION
XDH cloning: The XDH gene of C. capitata shares
considerable nucleotide and amino acid sequence iden-
tity with XDH genes of other insect species (Figure 1B).
Comparison of the known, complete XDH nucleotide
sequences produces the phylogenetic tree in Figure 2.
Not surprisingly, the CcXDH protein sequence is most
closely related to other sequences from dipteran species
and less similar to the sequences of the two XDH genes
of the lepidopteran, B. mori. Given that the dipteran
XDHs are all single copy genes, their relationships
might reasonably be considered orthologous, or all de-
rived from a common ancestor gene by speciation in
the insect lineages. Further evidence for XDH orthology
was presented by Komoto et al. (1999), who noted that
upstream of BmXDH2 is a region homologous to
l(3)12s, a gene that is similarly located upstream of the
XDH genes of D. melanogaster, D. pseudoobscura, and D.
subobscura (Riley 1989; Dutton and Chovnick 1991;
Comeron and Aguade 1996). The apparent XDH du-
plication event within B. mori thus occurred after the
divergence of the dipteran and lepidopteran orders
(Komoto et al. 1999).
As indicated for the white genes of several insect spe-
cies (Gomulski et al. 2001), the evolutionary relation-
ships of the reported species are also reflected by the
conservation of intron positions within the XDH gene
(Komoto et al. 1999). For example, the intron positions
A, B, F, and G of the CcXDH gene are conserved when
compared with the intron positions for several other
insect species (Komoto et al. 1999). It is interesting to
note that the medfly gene lacks introns B, C, and E,
introns that may have been lost during the evolution of
the XDH gene. This is reminiscent of the loss of introns
that has been suggested for introns B, C, E, and F of
the XDH gene of D. melanogaster (Komoto et al. 1999).
Alternatively, those introns may have been gained later
1652 R. J. Pitts and L. J. Zwiebel
in XDH evolution and therefore persist in the genes of
B. mori and mammals (Komoto et al. 1999). Recent evi-
dence supports the introns-gained hypothesis through the
comparison of several partial XDH sequences (Tarrio et
al. 1998).
We find 35 polymorphic nucleotides between our se-
quence and the partial sequence of 2085 bp reported
by Tarrio et al. (1998), which would lead to eight amino
acid substitutions. These differences probably reflect
XDH allelic differences within the same laboratory
strain, since both studies used Benakeion DNA, a fact
that is not surprising given that several XDH allozymes
have already been described in the medfly (Malacrida
et al. 1992). Furthermore, multiple XDH alleles have
been identified in a laboratory population of C. vicina
(Rocher-Chambonnet et al. 1987; Houde et al. 1989)
and a high level of heterozygosity has been observed at
the XDH locus in D. pseudoobscura (Singh et al. 1976).
The XDH gene of C. capitata was shown to encode
an active XDH enzyme by its ability to rescue the eye
color phenotype of the D. melanogaster mutant, ry506,
which lacks any detectable XDH activity (for review see
Chovnick et al. 1990). Homozygous ry506 Drosophila that
carried a heat-shock-driven CcXDH cDNA had normal,
bright red eyes, while those lacking the CcXDH had the
deep red eyes characteristic of the rosy mutant (Figure
3, C and D) even under heat-shock conditions. This
result demonstrates that the CcXDH cDNA encodes a
functional enzyme that is also biologically active in het-
erologous species separated by over 100 million years of
evolution (Beverly and Wilson 1984). Heterologous
rescue of the Drosophila rosy mutant has been previously
shown using a chimerical XDH containing the C-termi-
nal portion of the C. vicina XDH gene and an N-terminal
Figure 5.—XDH enzyme activity. (A)
Extracts from wild-type and rosy mutant
medflies display XDH enzyme activity
when hypoxanthine or xanthine are
used as a substrates. Similarly prepared
Drosophila extracts serve as controls.
(B) The chemical reactions catalyzed by
XDH include the conversion of hypo-
xanthine to xanthine and xanthine to
uric acid.
portion of the D. melanogaster XDH including the 5⬘
UTR (Tiveron et al. 1991). These results indicate that
CcXDH has the necessary functionality for use as a
marker for germline transformation within the medfly.
rosy-like mutant: The most likely C. capitata XDH mu-
tant isolated to date, rosy, displays a deep red eye color
and a sensitivity to purine supplemented growth media,
both characteristic of Drosophila rosy (i.e., XDH) mu-
tants (Saul 1982). Although red drosopterin pigments
have not yet been identified in wild-type medflies
(Ziegler and Feron 1965), it remains unclear as to
whether or not they are present in medfly eyes. Indeed,
the existence of the medfly rosy mutant provides strong,
albeit circumstantial, evidence for their presence. Fur-
ther study of eye pigments in the medfly may also lend
insight into the nature of the medfly rosy mutant. For
example, because the Drosophila rosy mutant specifi-
cally lacks isoxanthopterin and accumulates 2-amino-
4-hydroxyterin, the pteridine product and substrate of
the XDH enzyme, respectively (Reaume et al. 1991), a
similar pattern might be expected to occur in rosy
medflies. It is also formally possible that the medfly
lacks drosopterin pigments and the rosy phenotype is
the result of a defect in a gene unrelated to XDH.
Interestingly, the medfly XDH gene has been geneti-
cally mapped by allozyme analysis to the same position as
the medfly rosy mutant on linkage group D, the genetic
element that has subsequently been renamed chromo-
some 2 (Saul and Rossler 1984; Saul 1986; Mala-
crida et al. 1990, 1992). In fact, cytological mapping by
in situ hybridization to polytene chromosomes of the
CcXDH cDNA reported here has further refined its loca-
tion to section 4C of the long arm of chromosome 2
(A. R. Malacrida and C. Torti, personal communica-
 XDH cDNA of C. capitata
tion). As more genetic markers become available in
medfly, finer mapping studies of rosy should be possible,
either confirming or rejecting its potential correlation
to XDH. Given the similar phenotypes of medfly rosy
and Drosophila rosy, as well as the correlation of CcXDH
with the rosy mutant on the medfly genetic map, we
examined the medfly rosy mutant for potential abnor-
malities at the XDH locus.
While we were unable to detect gross DNA differences
such as large deletions, insertions, or chromosomal re-
arrangements between the wild type and rosy mutant
medflies in our genomic analyses (Figure 4A), we could
not eliminate the possibility that point mutations, small
deletions, or small insertions may lie within the XDH
coding region of the rosy mutant. This is especially rele-
vant since the rosy mutant was generated by formalde-
hyde treatment of medfly eggs, a process thought to
cause small DNA mutations (Saul 1982). To further
compare the XDH gene products, we carried out a re-
verse transcription/PCR analysis of wild-type and rosy
RNAs. In these studies, indistinguishably sized XDH
transcripts could be amplified from several develop-
mental stages of wild-type medflies as well as rosy pupae
(Figure 4B). These data show a similar developmental
pattern of XDH transcript expression in wild-type
medflies as in wild-type Drosophila (Covington et al.
1984). Furthermore, the XDH expression that was de-
tected in rosy pupae occurs at a time that is relevant for
eye color development in Drosophila and is therefore
likely to be equally important for the medfly (Barrett
and Davidson 1975). Lastly, the primers used for RT-
PCR expression studies were located near the 3⬘ end of
the gene and as such indicate that the transcript is likely
to be full length and stable. Taken together, these data
suggest that the rosy mutant might not be the result of
the alteration or loss of XDH transcripts. Again, as with
the DNA analysis, these data neither rule out the possi-
bility that small, mutagenic changes exist with the
CcXDH mRNA and are responsible for the mutant phe-
notype, nor eliminate the possibility that the transcript is
otherwise improperly translated but the protein remains
functional in vitro.
In the absence of a functional antiserum against
medfly XDH that might be used to directly assay protein
levels, we attempted to address the possibility that the
medfly rosy mutant lacks or has aberrant levels of XDH
enzymatic activity. In these studies, we examined the
ability of crude extracts from wild type as well as rosy
medflies to reduce xanthine or hypoxanthine in vitro
and to produce a colorimetric reaction product on cellu-
lose acetate gels. Importantly, we were able to take ad-
vantage of the availability of well-characterized Drosoph-
ila wild-type and ry506 strains for the preparation of
extracts to serve as positive and negative controls for
XDH activity, respectively. These data (Figure 5A)
clearly demonstrate that the medfly rosy mutant retains
considerable, if not wild-type, levels of XDH enzymatic
1653
activity in vitro. Whether or not the mutant has XDH
activity in vivo has not been directly examined. In Dro-
sophila, XDH is synthesized in the fat bodies sur-
rounding the eye and must be transported into the
eye for normal pigmentation to develop (Barrett and
Davidson 1975; Reaume et al. 1989, 1991). It is possible
that the medfly rosy mutant may produce an enzyme in
vivo that is either inactive or not expressed in the proper
tissues or at the proper time during development. It is
also formally possible that our assay system using crude
extracts cannot detect specific mutations, particularly
point mutations, within the XDH enzyme that would
alter, but not eliminate, XDH activity or localization.
Several detailed Drosophila studies have been carried
out that define functional domains of the XDH peptide
where known single amino acid changes in rosy mutants
affect XDH activity (Hughes et al. 1992; Hughes 1992;
Doyle et al. 1996). In those experiments, extracts from
some rosy mutants, such as G800E and G1011E, show
relatively high levels of in vitro activity in some assays,
especially following mild oxidation (Hughes et al. 1992;
Hughes 1992; Doyle et al. 1996). Therefore we cannot
rule out the possibility that the rosy mutant of medfly
may be the result of a point mutation in XDH that is
not discernible in our enzyme activity assays or that mild
oxidation of XDH occurs during the extraction process.
On the basis of our examinations of the CcXDH locus
at the levels of DNA, RNA, and enzyme activity, we
cannot support, nor conclusively rule out, the possibility
that the medfly rosy phenotype is caused by a mutation
at the CcXDH locus. Definitive evidence would best be
obtained by performing medfly transformation rescue
experiments using rosy medflies and an appropriate ex-
pression construct with the functional, wild-type cDNA
reported here along with either the Minos or piggyBac
vectors that have been previously used in the generation
of transgenic medflies (Loukeris et al. 1995; Handler et
al. 1998). However, because of the absolute quarantine
against live medflies in the continental United States,
we are prevented from carrying out this final test. Such
an experiment would help define the potential of the
rosy mutant to be used in combination with the wild-
type cDNA as a marker for germline transformation
experiments in medfly. Aside from its proposed func-
tion as a visible marker, the wild-type CcXDH may be
useful as a selectable marker if it can restore purine
resistance alone in the medfly rosy mutant and may
thereby contribute to population control strategies.
We thank Dr. F. C. Kafatos and the entire faculty and staff at
The European Molecular Biology Laboratory (Heidelberg, Germany)
where this work was initiated. We are very grateful to Dr. Anna Mala-
crida and Cristina Torti for providing unpublished XDH in situ map-
ping information as well as helpful discussions of XDH function
(Universita di Pavia, Italy). We thank Dr. C. S. Thummel (Univer-
sity of Utah, Salt Lake City) for Drosophila transformation vector
pP{CaSpeR-hs/act}, Dr. N. H. Brown (Wellcome/CRC Institute, Cam-
bridge, UK) in whose laboratory the Drosophila transformations were
carried out, and Dr. D. E. McCauley (Department of Biology, Vander-
1654 R. J. Pitts and L. J. Zwiebel
bilt University) for instruction in the use of cellulose acetate gels.
Special thanks to Dr. D. J. Funk (Department of Biology, Vanderbilt
University) and Dr. L. M. Gomulski (Universita di Pavia) for critical
review of the manuscript with particular regard to XDH phylogeny.
We also thank Richie Lin and Clay Ross for technical support as well
as A. N. Fox, C. E. Merrill, and other members of the Zwiebel labora-
tory for comments on the manuscript. This research was supported
by the U.S. Department of Agriculture-CSRS (92-37302-8237) to L.J.Z.
and the National Science Foundation (US)-NATO fellowship
(9255297) to L.J.Z.
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Communicating editor: T. C. Kaufman